ES2663304T3 - Means for the specific detection of resistant microorganisms - Google Patents

Abstract

Use of a culture medium to distinguish: * a first group of E. coli BLSE bacteria; * a second group of KESC BLSE bacteria; * a third group of bacteria not resistant to beta-lactamines; said culture medium comprising: * a first substrate that allows the detection of a beta-glucuronidase or beta-galactosidase enzyme activity; * a second substrate that allows the detection of a beta-glucosidase or tryptophanase or deaminase activity; * cefpodoxime; * cloxacillin.

Description

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DESCRIPTION

Means for the specific detection of resistant microorganisms

The field of the invention is that of microbiological analysis by biochemical route, and in particular of the detection and identification of microorganisms, such as, in particular, bacteria or yeasts.

The resistance of bacteria to antibiotics is a major public health problem. The resistance of infectious microorganisms to a treatment has developed at the same time as the anti-infective molecules and currently represents an important therapeutic obstacle. This resistance is a source of multiple problems, including laboratory detection difficulties, limited treatment options and a detrimental impact on the clinical outcome.

In particular, the rapid and irrepressible increase in resistance of pathogenic bacteria, during these last 20 years, represents one of the main current problems of medicine. Infections caused by these organisms are the origin of the lengthening of hospitalization duration and are associated with high morbidity and mortality rates, after therapeutic failures.

Several resistance mechanisms may be involved simultaneously in a bacterial strain. They are generally classified into 3 categories: deficiency of the penetration of the antibiotic in the bacterium, inactivation or excretion of the antibiotic by bacterial enzymatic systems and lack of affinity between the bacterial target and the antibiotic.

Enzymatic inactivation is the most frequent mechanism of resistance acquired in terms of number of species and antibiotics in question. Thus, class C chromosomal cephalosporinases are currently one of the predominant resistance mechanisms of gram-negative bacteria, the bacteria expressing such cephalosporin-resistant enzymes. Likewise, p-lactamases are enzymes expressed by certain bacteria, capable of hydrolyzing the C-N bond of the p-lactam nucleus, the base structure of the antibiotics of the p-lactamines family, to give a microbiologically inactive product. Several p-lactamase (IBL) inhibitors, such as clavulanic acid (AC), tazobactam and sulbactam, have been developed to increase antimicrobial activity and widen the spectrum of the associated p-lactamines. Acting as a suicide substrate for p-lactamases, they prevent the enzymatic degradation of antibiotics and allow them to become effective against initially resistant bacteria. However, due to the persistent exposure of the strains to antibiotic pressure, the bacteria express their ability to adapt by the continuous and dynamic production of p-lactamases, evolving at the same time as the development of the new molecules.

Gram-negative bacteria producing high-level class C chromosomal cephalosporinases (referred to as Case HN bacteria), as well as gram-negative bacteria producing broad-spectrum p-lactamases (then talking about BLSE bacteria) have become thus in an increasing threat, in particular because the number of bacterial species in question increases. The Case HN and BLSE bacteria are resistant to treatments based on penicillins and cephalosporins of 1st and 2nd generation, but also of 3rd generation cephalosporins (C3G) (cefotaxime CTX, ceftazidime CAZ, cefpodoxime CPD, ceftriazone CRO) and monobactams (ATM aztreonam ). In contrast, 7a-methoxycephalosporins (cephamycins: cefoxitin, cefotetan) and carbapenems (imipenemo, ertapenemo meropenemo) generally retain their activity. BLSEs are inhibited by p-lactamase inhibitors (IBL), which allows them to be differentiated from other cephalosporinases.

These bacteria generally thus express resistance to several treatments simultaneously, which poses difficulties in installing a relevant treatment and avoiding therapeutic failures. An Escherichia coli bacteria can thus be Case HN and BLSE. In addition, since BLSE positive enterobacteria tend to spread resistance by clonal transmission of strains or conjugative plasmid transfer, they represent a problem for infection control. In most studies, Escherichia coli and Klebsiella pneumoniae remain the most common BLSE producing species. But, for some years now, the BLSE have greatly expanded their panel of host species. Indeed, numerous species of non-fermenting gram-negative enterobacteria and bacilli (such as Pseudomonas aeruginosa) have also been identified as producers of BLSE.

In addition to these BLSE bacteria, Staphylococcus aureus bacteria can also be mentioned, which are also pathogenic bacteria that develop numerous resistance mechanisms, such as a resistance to methicillin, penicillin, tetracycline, erythromycin, vancomycin. Enterococcus faecium is another multiresistant bacteria found in a hospital environment, which can be resistant to penicillin, vancomycin and linezolid. Mycobacterium tuberculosis is usually resistant to isoniazid and rifampicin. Other pathogens offer certain resistance such as Salmonella, Campylobacter and Streptococcus.

Therefore, it becomes indispensable, from a public health point of view, to identify as quickly as possible such microorganisms, and such resistance mechanisms.

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In general, the search for microorganisms resistant to a treatment is carried out according to the following stages

1. extraction of a biological sample capable of containing said microorganisms,

2. sowing and incubation of a culture medium (18 to 48h) to induce exponential growth of microorganisms,

3. location on the culture media of colonies of potentially significant microorganisms,

4. characterization of the species of microorganism,

5. Identification of the resistance mechanisms of the microorganisms analyzed, their biological significance and eventually the adapted therapy.

This succession of stages implies an important amount of time between the extraction of the sample capable of containing microorganisms and the prescription of a suitable treatment for the patient. In addition, the user must generally manually carry out stages of transfer of microorganisms from a first medium to a second medium, which can cause problems, in particular contamination, but also risks to the health of the manipulator.

As an example, to detect the presence of broad-spectrum beta-lactamases (BLSE) in strains of Escherichia coli and Klebsiella pneumoniae, a diffusion technique can be used as described in the Jacoby & Han publication (J Clin Microbiol 34 (4): 908-11, 1996) which does not, however, give any information on the identification of the strains tested: it can be determined whether the bacterium is a BLSE-producing bacterium or not, but it cannot be distinguished if such a bacterium is an Escherichia coli or a Klebsiella pneumoniae.

Metabolic substrates are also used to detect the presence of BLSE or Case Hn. For this, the AES laboratories propose a medium in a bi-box that associates a Drigalski medium with cefotaxime and a MacConkey medium with Ceftazidime. Drigalski and MacConkey media allow to reveal the acidification of lactose, metabolism present in a large number of species of enterobacteria. However, such means only distinguishes resistant bacteria from non-resistant bacteria, but it does not distinguish bacteria that express a BLSE from those that express a Case Hn. This medium also does not allow the identification of particular species of bacteria, and does not allow, for example, to distinguish E. coli bacteria from K. pneumoniae bacteria.

In the case of the detection of resistance mechanisms other than BLSE, patent application EP0954560 can be cited, which refers to the search for vancomycin-resistant enterococci, combining Vancomycin with a chromogenic medium that reveals two enzymatic activities ( P-glucosidase and pyrrolidonyl arylamidase). However, this chromogenic medium allows to determine only if the vancomycin resistant strains belong or not to the Enterococcus genus, but it does not allow to identify the species or the resistance mechanisms involved, in particular if it is an acquired or wild resistance.

Thus, the characterization of a species of microorganism, and then the identification of its resistance to a treatment, is long and annoying. If the laboratory provides the clinician with a positive detection while the isolate is actually devoid of resistant microorganisms, this can lead to useless and inappropriate treatment. Conversely, not communicating a positive detection that will be confirmed after delays the implantation of the isolation of the patient (and possibly an appropriate therapy) in one day. This shows the need for a fast and reliable confirmation test.

The present invention therefore aims to improve the state of the art by presenting a new diagnostic tool that allows for a gain in time, reliability and relevance in the therapy used. Our invention allows, in a single stage, to identify the species of microorganisms present in a sample, to determine their resistance mechanism in order to propose a suitable treatment for each patient. This invention is particularly suitable for discriminating different species of microorganisms, which have different resistance mechanisms to different treatments, but all susceptible to being present in the same sample.

Before going further in the description of the invention, the following definitions are given in order to facilitate the understanding of the invention:

By culture means means comprising all the necessary elements for the survival and / or growth of microorganisms. The culture medium according to the invention may contain other possible additives, such as: peptones, one or more carbohydrate growth factors, one or more selective agents, buffers, one or more gelling agents, etc. This culture medium can be presented in the form of liquid, gel ready for use, that is, ready for inoculation in a tube, bottle, or on a Petri dish.

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Within the meaning of the present invention, the term "microorganism" covers bacteria, gram positive or gram negative, yeasts and, more generally, generally unicellular organisms, invisible to the naked eye, which can be multiplied and manipulated in the laboratory.

As gram-negative bacteria, bacteria of the following genera can be cited: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Branhamekholder, Neisseria, Buria , Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Pasteurella, Providencia, and Legionella.

As gram-positive bacteria, bacteria of the following genera can be cited: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Listera, Clostridium, Gardnerella, Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria and Corynebacteria. As yeasts, yeasts of the following genera can be cited: Candida, Cryptococcus, Saccharomyces and Trichosporon.

By biological sample, a clinical sample is understood, coming from an extraction of biological liquid, or a food sample, coming from any type of food. This sample can thus be liquid or solid and a clinical sample of blood, plasma, urine, feces, nose, throat, skin, wounds, cephalo-spinal fluid, a food sample of water, drinks such as milk, fruit juice; yogurt, meat, eggs, vegetables, mayonnaise, cheese; fish, etc., a food sample from an animal feed, such as in particular a sample from animal meal.

Resistance mechanisms are understood as any type of device that allows a microorganism to make a partial or totally inoperative treatment on said microorganism, guaranteeing its survival. The resistance mechanisms are generally classified into 3 categories: deficiency in the penetration of the antibiotic into the bacteria, inactivation or excretion of the antibiotic by bacterial enzyme systems and affinity deficiency between the bacterial target and the antibiotic.

By way of indication, resistance mechanisms related to the expression of an enzyme belonging to the group of broad-spectrum p-lactamases can be cited in particular; of an enzyme belonging to the group of high level class C chromosomal cephalosporinases; the mechanisms of resistance to glycopeptides, preferably developed by bacteria belonging to the genus Enterococcus.

The mechanisms of resistance to methicillin, penicillin, tetracycline, erythromycin, vancomycin will also be cited when the microorganism is a Staphylococcus aureus bacteria.

The mechanisms of resistance to penicillin, vancomycin and linezodil will also be cited when the microorganism is an Enterococcus faecium bacterium.

The mechanisms of resistance to amphotericin B or antifungals of the azolate family will also be cited when the microorganism is a yeast.

The mechanisms of resistance to isoniazid and rifampicin will finally be cited when the microorganism is a Mycobacterium tuberculosis bacterium.

By treatment, a treatment is understood to be able to prevent or reduce the growth of microorganisms from a patient. This treatment may in particular comprise antimicrobial compounds, such as antibiotics, such as penicillins, classical cephalosporins, broad-spectrum cephalosporins, monobactams, glycopeptides, aminosides or such as antifungal or resistance-inhibiting compounds. It is pointed out that this treatment may also include the isolation of the patient, which prevents the spread of the microorganism in other patients.

By substrate that allows the detection of an enzymatic or metabolic activity, any molecule capable of directly or indirectly generating a detectable signal due to an enzymatic or metabolic activity of the microorganism is understood.

When this activity is an enzymatic activity, it is then referred to as an enzyme substrate. By enzymatic substrate, any substrate that can be hydrolyzed by an enzyme in a product that allows the detection, directly or indirectly, of a microorganism is understood. This substrate comprises in particular a first specific part of the enzymatic activity to be disclosed and a second part that acts as a marker, hereinafter referred to as the labeling part. This marking part can be chromogenic, fluorogenic, luminescent, etc. As a chromogenic substrate, very suitable for solid supports (filter, gel, electrophoresis gel), it is possible to mention in particular substrates based on indoxyl and their derivatives, and substrates based on hydroxyquinoline or esculetin and their derivatives, which allow the detection of osidase and esterase activities. Mention may also be made of nitrophenol and nitroanaline-based substrates and derivatives, which allow the detection of osidases and esterases activities in the case of nitrophenol-based substrates, and of peptidases in the case of substrates based on

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nitroanaline Finally, the substrates based on naphthol and naphthylamine and their derivatives, which allow the detection of osidases and esterases by means of naphthol, and peptidases by means of naphthylamine can be mentioned. This substrate may in particular, but not limited to, allow the detection of an enzymatic activity such as the activity of an osidase, peptidase, esterase, etc. The enzyme substrate can also be a natural substrate whose hydrolysis product is detected directly or indirectly. As a natural substrate, Tryptophan can be cited in particular to detect a tryptophanase or deaminase activity, a cyclic amino acid (tryptophan, phenylalanine, histidine, tyrosine) to detect a deaminase activity, phosphatidyl inositol to detect a phospholipase activity, etc.

When this activity is a metabolic activity, the substrate is then a metabolic substrate, such as a carbon or nitrogen source, coupled to an indicator that produces a coloration in the presence of one of the metabolism products.

According to a preferred embodiment of the invention, said first and / or second enzymatic or metabolic activity is an enzymatic activity, preferably selected from the enzymatic activities: beta-glucosidase, deaminase, beta-glucuronidase, beta-galactosidase, alpha-glucosidase, alpha-galactosidase, hexosaminidase, N-acetyl-hexosaminidase, phosphatase, esterase, aminopeptidase.

For example, to detect E. coli, the activity beta-glucuronidase or p-galactosidase or tryptophanase or deaminase is preferably used; to detect Proteus, the deaminase activity is preferably used, to detect the enterococci, the beta-glucosidase activity is preferably used. For Candida albicans hexosaminidase is preferred, for Listeria monocytogenes phospholipase, for salmonellae, esterase, for Pseudomonas aeruginosa esterase or p-alanine aminopeptidase, for Staphylococcus aureus phosphatase or alpha-glucosidase.

By marker that allows the differentiation of two groups of microorganisms, a compound is understood that does not have the same properties in a first and a second group. This compound can thus be:

* a specific substrate

* a resistance mechanism inhibitor, which then allows to inhibit the growth of organisms that develop a particular resistance, without discrimination of the species of microorganisms.

In the case of the use of a specific substrate, the beta-glucuronidase, or beta-galactosidase or tryptophanaseou deaminase activity is preferably used to detect E. coli, to detect Proteus the deaminase activity is preferably used, to detect the enterococci, preferably uses the beta-glucosidase activity. For Candida albicans, hexosaminidase is preferred, for Listeria monocytogenes phospholipase, for salmonellae esterase, for Pseudomonas aeruginosa esterase or p-alanine aminopeptidase, for Staphylococcus aureus phosphatase or alpha-glucosidase.

In the case of the use of a resistance mechanism inhibitor, it is preferably used

* clavulanic acid, tazobactam or sulbactam when the first group and (or the second group comprises a mechanism of resistance induced by the expression of p-lactamases. The concentration of clavulanic acid in the medium is then preferably between 0.05 and 32 mg / l, preferably between 0.1 and 8 mg / l and even more preferably between 0.25 and 6 mg / l.

* cloxacillin or dicloxacillin when the first group and / or the second group comprises a mechanism of resistance induced by the expression of cephalosporinases

A taxon means a group of microorganisms that have a taxonomic unit. A taxon can be a family, a genus, a set of genera, a species, a set of species, a sub-species. For indicative purposes, the enterobacteria, Klebsiella, Escherichia, Enterobacter, Citrobacter, Serratia, KESC (Klebsiella, Enterobacter, Serratia, Citrobacter), Proteeae, Proteus, Morganella, Pseudomonas, Staphylococcus, Streptococcus, Enterobaccus, Candida Escherichia coli O157: H7, Klebsiella pneumoniae, Citrobacter freundii, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, staphylococci to coagulase negative, Candida albicans, Candida glabrata, Candida krusei, Candida luse, Candida lusiae

By antimicrobial, any compound capable of preventing or slowing the growth of a microorganism is understood. This compound can be an antibiotic or an antifungal.

By antibiotic, any compound capable of preventing or slowing the growth of a bacterium is understood. By way of indication, the antibiotics cefotaxime, ceftazidime, ceftriaxon, cefpodoxime, aztreonam, vancomycin, tobramycin, ciprofloxacin can be cited in particular.

By antifungal, is meant any compound likely to prevent or slow the growth of a yeast

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or of a mold. By way of indication, one can cite in particular amphotericin B, fluconazole, itraconazole, voriconazole, cycloheximide.

According to a preferred embodiment of the invention, when the antibiotic is

* cefotaxime, the concentration of cefotaxime in the medium is preferably between 0.25 and 8 mg / l, preferably between 1 and 2 mg / l.

* Ceftazidime, the concentration of ceftazidime in the medium is preferably between 0.25 and 8 mg / l, preferably between 2 and 2.5 mg / l.

* Ceftriaxone, the concentration of ceftriaxone in the medium is preferably between 0.25 and 8 mg / l, preferably between 1 and 2.5 mg / l.

* cefpodoxime, the concentration of cefpodoxime in the medium is preferably between 0.1 and 32 mg / l, preferably between 0.75 and 10 mg / l and even more preferably between 1 and 6 mg / l.

* Aztreonam, the concentration of aztreonam in the medium is preferably between 0.1 and 8 mg / l, preferably between 0.75 and 1.5 mg / l.

According to a particular embodiment of the invention, the medium comprises a combination of at least two antibiotics. Preferably, the combination of at least two antibiotics comprises cefotaxime and ceftazidime.

Whatever the embodiment of the invention, the medium may further comprise a dye. By way of indication, Evans blue, neutral red, sheep's blood, horse's blood, an opacifier such as titanium oxide, nitroanaline, malachite green, bright green, etc. can be cited as a colorant.

All means may further comprise in order to increase their sensitivity.

* at least one active antimicrobial against gram-positive bacteria, such as in particular linezolide or vancomycin,

* at least one active antimicrobial against yeasts, such as in particular voriconazole or amphotericin B.

The invention relates to the use of a culture medium to distinguish at least 3 groups of microorganisms in a biological sample comprising:

* a first group of microorganisms, which belongs to a first taxon of microorganisms and which comprises at least one treatment resistance mechanism,

* a second group of microorganisms, which belongs to a second taxon of microorganisms, different from said first taxon, but comprising at least one treatment resistance mechanism, identical to that of the first group

* a third group of microorganisms, not resistant to said treatment comprising said culture medium

to. at least a first substrate that allows the detection of at least a first enzymatic or metabolic activity of said first group of microorganisms

b. at least one marker that allows the differentiation of the first group of microorganisms and the second group of microorganisms, said marker being a substrate that allows the detection of at least one enzymatic or metabolic activity of said second group of microorganisms

C. at least one antimicrobial, active on said third group of microorganisms

This embodiment of the invention allows to distinguish in the same sample a first and a second group comprising different species or different taxa of microorganisms, but each of the two groups being resistant to the same treatment.

This embodiment of the invention thus allows, for example, to distinguish, in the same sample, a first group comprising E. coli BLSE bacteria, and a second group comprising KESC BLSE bacteria.

The invention relates to a culture medium comprising:

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* a first substrate that allows the detection of a beta glucuronidase or beta galactosidase enzyme activity, preferably 6-Chloro-3-indolyl-pD-glucuronide at a concentration between 25 and 750 mg / l, preferably between 40 and 300 mg / l 5-Bromo-6-chloro-3-indolyl-pD-galactoside at a concentration between 25 and 500 mg / l, preferably between 40 and 150 mg / l

- a second substrate that allows the detection of a beta glucosidase activity, preferably 5-Bromo-4-chloro-3-indolyl-pD-glucoside at a concentration between 25 and 500 mg / l, preferably between 40 and 250 mg / tryptophanase or deaminase, preferably tryptophan at a concentration between 50 and 5000 mg / l, preferably between 250 and 2000 mg / l

- an antimicrobial which is an antibiotic, preferably cefpodoxime at a concentration between 0.5 and 32 mg / l, preferably between 0.75 and 10 mg / l and even more preferably between 1 and 6 mg / l and cloxacillin at a concentration between 10 and 2000 mg / l, preferably between 50 and 500 mg / l

When the antibiotic is cefpodoxime, this medium is preferably used to distinguish:

* a first group of E. coli BLSE bacteria

* a second group of KESC BLSE bacteria

* a third group of bacteria, not resistant to beta lactamines. The invention also relates to a culture medium comprising:

* a first substrate that allows the detection of a beta glucuronidase enzyme activity, preferably 6- Chloro-3-indolyl-pD-glucuronide at a concentration between 25 and 750 mg / l, preferably between 40 and 300 mg / l beta galactosidase, preferably 5-Bromo-6-chloro-3-indolyl-pD-galactoside at a concentration between 25 and 500 mg / l, preferably between 40 and 150 mg / l

* a second substrate that allows the detection of a beta glucosidase activity, preferably 5-Bromo-4-chloro-3-indolyl-pD-glucoside at a concentration between 25 and 500 mg / l, preferably between 40 and 250 mg / tryptophanase or deaminase, preferably tryptophan at a concentration between 50 and 5000 mg / l, preferably between 250 and 2000 mg / l

* two antimicrobials, which are preferably two antibiotics, preferably cefpodoxime at a concentration between 0.1 and 32 mg / l, preferably between 0.25 and 10 mg / l and even more preferably between 0.5 and 4 mg / l and aztreonam at a concentration between 0.1 and 8 mg / l, preferably between 0.5 and 1.5 mg / l

This medium is preferably used to distinguish:

* a first group of E. coli BLSE or Case NH bacteria

* a second group of KESC BLSE or Case NH bacteria

* a third group of bacteria, not resistant to beta-lactamines and / or cephalosporins The invention also relates to a culture medium comprising:

* an antimicrobial, preferably an antibiotic such as cloxacillin

* a resistance mechanism inhibitor, such as preferably a 3rd generation cephalosporin selected from cefotaxime, ceftazidime, cefpodoxime, ceftriaxone.

This medium is also preferably used to detect BLSE bacteria.

The embodiment of the invention described above is not limited to the distinction of 3 groups of microorganisms, but may allow the distinction of 4, 5 or even more groups of microorganisms. It is then necessary to add identification markers between the different groups in the middle.

For this reason, the invention also relates to a culture medium comprising:

* a first substrate that allows the detection of a beta glucuronidase enzyme activity, preferably 6- Chloro-3-indolyl-pD-glucuronide at a concentration between 25 and 750 mg / l, preferably between 40 and 300 mg / l beta galactosidase, preferably 5-Bromo-6-chloro-3-indolyl-pD-galactoside at a concentration between 25 and 500 mg / l, preferably between 40 and 150m / l

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* a second substrate that allows the detection of a beta glucosidase, preferably 5-Bromo-4-chloro-3- indolyl-p-D-glucoside at a concentration between 25 and 500 mg / l, preferably between 40 and 250 mg / l

* a combination of antimicrobial, preferably a combination of antibiotics such as

° cefpodoxime at a concentration between 0.5 and 32 mg / l, preferably between 0.75 and 10 mg / l and even more preferably between 1 and 6 mg / l,

° cloxacillin at a concentration between 10 and 2000 mg / l, preferably between 50 and 500 mg / l,

° vancomycin at a concentration between 0.5 and 128 mg / l, preferably between 2 and 32 mg / l et-

° amp B at a concentration between 0.5 and 64 mg / l, preferably between 1 and 16 mg / l, even more preferably between 1 and 8 mg / l

* a third substrate that allows the detection of a deaminase activity such as histidine, phenylalanine, tryptophan or tyrosine at a concentration between 50 and 5000 mg / l, preferably between 250 and 2000 mg / l.

This medium may further comprise a fifth antibiotic that is cefsulodine, at a concentration between 0.5 and 64 mg / l, preferably between 1 and 16 mg / l.

This medium is preferably used to distinguish:

* a first group of E. coli BLSE bacteria

* a second group of KESC BLSE bacteria

* a third group of bacteria, not resistant to beta-lactamines and / or cephalosporins - a fourth group of Proteeae BLSE bacteria

By using an adapted combination of antimicrobials, it is possible to distinguish not only three groups of microorganisms but also 4, 5 or even more groups of microorganisms.

The following examples are given for explanatory purposes and do not have any limiting character. They will allow a better understanding of the invention.

EXAMPLE 1

The example below is based on the phenotypic detection of BLSE using the reduction in susceptibility of these strains to antibiotics and their sensitivity to associations with p-lactamase inhibitors. For this, a bi-box based on CPS ID 3 (chromogenic medium for the detection of microorganisms in the urine and sold by bioMérieux under reference 43541) has been used with a semi-gel containing an antibiotic and a semi-gel It contains an antibiotic association - p-lactamase inhibitor.

1. Choice of strains

In the field of manipulations carried out, for the evaluation of the activity of active antibiotics on gram-negative bacilli, different species of enterobacteria (Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis) and of non-fermenting gram-negative bacilli (Pseudomonas aeruginosa) capable of producing BLSE. In the trials, BLSE positive strains, high-level cephalosporinase producing strains (Case HN) and wild strains are compared.

For manipulations referring to active antibiotics on gram-positive bacteria, gram-positive coconut strains have been tested (Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Streptococcus agalactia gram degalactia bagactia degalactia gram) -positives (Lactobacillus spp).

For trials aimed at evaluating the activity of antifungals, different strains of yeasts have been used (Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis, Saccharomyces cerevisiae, Geotrichum capitatum).

2. Media preparation

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The medium used was a CPS ID3 medium (reference 43541) which further comprises at least one antibiotic and at least one resistance mechanism inhibitor.

The composition of the media tested was as follows:

 Chromogenic substrate Antibiotic Resistance inhibitor

 Medium A

 6-Chloro-3-indolyl-p-D-glucuronide 5-Bromo-4-chloro-3-indolyl-p-D-glycoside Tryptophan Total FeCla: 1.73g / l Cefotaxime 1 mg / l

 Medium B

 6-Chloro-3-indolyl-p-D-glucuronide 5-Bromo-4-chloro-3-indolyl-p-D-glycoside Tryptophan FeCls Total: 1.73g / l Ceftazidime 1.5 mg / l Clavulanic Ac 0.25 mg / l

Osmotic water is added and the whole is homogenized and melted in a water bath at 100 ° C. The base medium is distributed in jars, in a number that corresponds to the total number of media to be tested during handling. The bottles are then autoclaved for 15 minutes at 121 ° C. The media are brought back and kept in superfusion at 55 + 3 ° C to the water bath, in order to sterilely add the thermolabile additives (previously sterilized by filtration over 0.22 µm).

The media are then poured into bi-boxes 90 mm in diameter (ie approximately 9.5 ml / semi-box) and left on a flat surface so that they can recover the dough. Then the surface of the twins is dried under a laminar flow hood for 30 minutes.

3. Media inoculation

From a 24-hour preculture at 36 ° C + 2 ° C in an aerobic atmosphere on TSA medium, an inoculum of 0.5 McF is prepared in physiological water, then 1 µl of this suspension is transferred in 5 ml of physiological water . In order to obtain sufficiently numerous isolated colonies, a range of inoculums allowed to determine that the optimum amount of bacteria to be inoculated was 103 to 104 CFU / ml. The inoculation is carried out directly on the 2 semi-gelosa with the help of a sterile swab. The cultures are then incubated at 37 ° C in an aerobic atmosphere.

4. Media reading

The readings are made at 18 hours (+ 30 minutes), 24h (+ 1h) and 48h (+ 4h). The density and size of the colonies, the appearance, color and intensities of coloration of the mass and the isolated colonies have been observed, according to the following reading scales 1 to 3: 0: no growth; 0.1: trace of growth; 0.25: colonies with a diameter <0.5; 0.5: colonies 0.5 mm in diameter; 0.75: 0.5 mm <diameter <1 mm; 1: colonies of 1 mm in diameter; 1.25: 1 mm <diameter <1.5; 1.5; 1.5 mm diameter colonies; 2: 2 mm diameter colonies; 3: colonies with a diameter> 2 mm.

5. Results:

a) Antibiotic screening

The 5 antibiotics recommended by the NCCLS (Natinal Committee for Clinical Laboratory Standars) were tested. For each product tested, a range was made to determine the concentrations that allow inhibiting wild strains of the enterobacteria tested, without affecting the growth of BLSE positive strains or Case HN positive strains.

The concentrations chosen are classified in Table III below.

Table III Threshold values of antibiotic concentrations that allow an inhibition of wild strains of the enterobacteria tested, without affecting the growth of BLSE-positive strains under conditions

rehearsed

 Base value High value

 Cefotaxime

 1 mg / l 2 mg / l

 Ceftazidime

 2 mg / l 2.5 mg / l

 Ceftriaxone

 1 mg / l 2.5 mg / l

 Cefpodoxime

 2 mg / l 10 mg / l

 Aztreonam

 1 mg / l 1.5 mg / l

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The base value corresponds to the minimum concentration of the antibiotic necessary for the inhibition of wild strains of the enterobacteria tested. The high value corresponds to the maximum concentration of the antibiotic that can be used without affecting the growth of the tested BLSE strains tested.

At these concentrations, the separation of wild and resistant strains is satisfactory, and the expression of enzymatic activities on the CPS ID 3 medium.

In addition, it is noted that ceftazidime is the only antibiotic tested and used in the detection of BLSE that has shown an activity on wild strains of P. aeruginosa. A range has been made in order to define the minimum concentration that allows a total inhibition of these strains and the threshold value is 1.5 mg / l.

The addition of antibiotics had no influence on the expression of bacterial enzymatic activities on the chromogenic medium. The germ groups were also better separated and identified in control CPS ID 3 medium than on the media comprising antibiotics as described above.

b) B-lactamase inhibitor screening

Three inhibitors of B-lactamases (IBL) have been used, namely clavulanic acid, tazobactam and sulbactam. Each was subject to a range, in the presence of cefotaxime, in order to determine the optimal concentration to inhibit BLSE positive strains, without impeding the growth of Case HN strains.

Clavulanic acid appeared as the most effective IBL in the presence of cefotaxime. Tazobactam and sulbactam needed concentrations higher than 2 mg / l to inhibit BLSE positive strains, while clavulanic acid was more active at clearly lower concentrations and over a wide range of use (0.1 to 8 mg / l when used in association with cefotaxime).

Each antibiotic (cefotaximin, ceftazidimine, ceftriaxone, cefpodoxime, aztreonam) was tested in the presence of a range of clavulanic acid in order to define the most effective combinations. The combinations chosen are classified in table IV below.

Table IV Selected associations of antibiotics and clavulanic acid (AC), which inhibit the strains of enterobacteria tested BLSE-positive, but allow Case HN positive strains to grow.

 Cefotaxime

 2 mg / l + AC 0.1 mg / l

 Ceftazidime

 2.5 mg / l + AC 2 mg / l

 Ceftriaxone

 2 mg / l + AC 0.25 mg / l

 Cefpodoxime

 9 mg / l + AC 0.25 mg / l

 Aztreonam

 1 mg / l + AC 0.5 mg / l

The results obtained using a bi-box containing the antibiotic alone and on the other hand the antibiotic + clavulanic acid association are classified in Table V.

Table V

 Antibiotic only Antibiotic + clavulanic acid

 E. coli wild

 E. coli BLSE positive

 Pink colonies

 E. coli Case HN positive

 Pink colonies Pink colonies

 Wild proteeae

 Proteae BLSE positive

 Brown colonies

 Proteeae Case HN positive

 Brown colonies Brown colonies

 Wild kesc

 KESC BLSE positive

 Green colonies

 KESC Case HN positive

 Green colonies Green colonies

The addition of B-lactamase inhibitors had no influence on the expression of bacterial enzymatic activities on the chromogenic medium. The germ groups were also better separated and identified on control CPS ID 3 medium than on media comprising B-lactamase inhibitors.

c) Antibiotic combination

Taking into account the relative activities of antibiotics and clavulanic acid, they have been associated

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* cefotaxime (active antibiotic on BLSE positive strains in combination with the lowest concentration of clavulanic acid).

* ceftazidime (active antibiotic on wild strains of P. aeruginosa), and

* clavulanic acid,

in order to be able to inhibit on the one hand the wild strains (including those of the pyocyanic bacillus), and the BLSE positive strains of the enterobacteria tested by the addition of IBL.

Such an association has been tested on strains of P. aeruginosa, this species is naturally resistant to cefotaxime but sensitive to ceftazidime. By associating 1.5 mg / l of CAZ (ceftazidime), 1 mg / l of CTX (cefotaxime) and 0.25 mg / l of AC (clavulanic acid), all wild strains and BLSE positive strains of the enterobacteria tested are they inhibited and grew only the set of Case HN strains and BLSE positive strains of P. aeruginosa. It is a bi-box with, on the one hand, CAZ alone and, on the other hand, CTX plus clavulanic acid.

The addition of these antibiotic combinations had no influence on the expression of bacterial enzymatic activities on the chromogenic medium. The germ groups were as well separated and identified on CPS ID 3 control medium as on the media comprising such combinations of antibiotics.

d) Dye test

The medium according to the invention has also been made for use in a bi-box, one side of which contains an antibiotic, and the second another antibiotic or a combination of antibiotics. Since the same CPS ID 3 media base is used on both sides, these 2 sides were differentiated by the presence of a dye.

The dye tested, Evans blue, gives the medium a green color. The coloration allowed to easily differentiate the 2 sides of the bi-box, without affecting the fertility of the environment, nor hinder the reading of the enzymatic activities of the colonies. After having made a range of Evans blue, added before or after autoclaving, the chosen values were as follows:

-1.5 or 2 mg / l if the dye is added after autoclaving - between 2 and 5 mg / l if it is added before autoclaving.

e) Inhibition of Gram-positive bacteria

BLSEs are a mechanism of resistance to p-lactamines, found only in gram-negative bacilli, therefore it is appropriate to inhibit gram-positive bacteria in the medium. Two antibiotics, linezolide and vancomycin were used in the medium according to the invention to inhibit sensitive gram-positive bacteria.

For vancomycin, under the conditions tested, a concentration between 2 and 32 mg / l and in particular 2 to 5 mg / l allows the inhibition of sensitive gram-positive bacteria without interfering with the detection of BLSE bacteria.

For linezolid, under the conditions tested, a concentration between 2 and 64 mg / l and in particular 4 to 16 mg / l allows the inhibition of sensitive gram-positive bacteria without interfering with the detection of BLSE bacteria.

The inhibition of gram-positive bacteria allowed to improve the detection of BLSE bacteria in polymicrobial extractions and the specificity of their coloration.

f) Inhibition of yeasts

The medium according to the invention may also comprise antifungals in order to inhibit the eventual presence of yeasts that could grow on the medium and that could disturb the growth of germs.

Two antifungals have therefore been tested: voriconazole and amphotericin B.

For amphotericin B, under the conditions tested, a concentration between 1 and 32 mg / l and in particular 2 to 8 mg / l allows the inhibition of sensitive yeasts without interfering with the detection of BLSE bacteria.

For voriconazole, under the conditions tested, a concentration between 1 and 64 mg / l and in particular 4 to 16 mg / l allows the inhibition of sensitive yeasts without interfering with the detection of BLSE bacteria.

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The inhibition of yeasts allows to improve the detection of BLSE bacteria in polymicrobial extractions and the specificity of their coloration.

6. Conclusion

These results demonstrate that the medium according to the invention allows the isolation and presumed identification of the BLSE producing bacteria, differentiating them from the high level cephalosporinase producing strains. The use of a bi-box containing one cephalosporin alone on one side, and on the other a cephalosporin / clavulanic acid association, based on CPS ID 3, is particularly interesting.

Example 2

This second example is based on the phenotypic detection of BLSE using the reduction of antibiotic susceptibility and the sensitivity of Case HN to concentrations with tobramycin or cloxacillin or di-cloxacillin and presents the use of a cephalosporin mentioned above (CTX, CAZ, CPD, CRO, ATM) in combination with a compound that inhibits cephalosporinases (cloxacillin, di-cloxacillin and tobramycin). Such a medium allows the inhibition of bacteria that have a "natural" cephalosporinase, most of these having only a high level cephalosporinase (Case HN), while allowing the growth of BLSE bacteria.

1. Choice of strains

In the field of manipulations carried out, for the evaluation of the activity of active antibiotics on gram-negative bacilli, different species of enterobacteria (Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis) and gram-bacilli have been used non-fermenting negatives (Pseudomonas aeruginosa) susceptible to produce BLSE. In the trials, BLSE positive strains, high-level cephalosporinase producing strains (Case HN) and wild strains are compared.

2. Preparation of the medium

The medium used was a CPS ID3 medium (43541) which further comprises

* ceftazidime at 2.5 mg / l and tobramycin at 2 mg / l (medium A) or

* 2 mg / l ceftriaxone and 150 mg / l cloxacillin (medium B) or

* cefpodoxime at 2 mg / l and di-cloxacillin at a concentration between 500 and 1000 mg / l (medium C)

Osmotic water is added and the whole is homogenized and melted in a water bath at 100 ° C. The base medium is distributed in jars, in a number that corresponds to the total number of media to be tested during handling. The bottles are then autoclaved for 15 minutes at 121 ° C. The media are brought back and kept in superfusion at 55 + 3 ° C to the water bath, in order to sterilely add the thermolabile additives (previously sterilized by filtration over 0.22 µm).

The media are then poured into 35 mm diameter boxes and left on a flat surface so that they can recover the dough. Then the surface of the twins is dried under a laminar flow hood for 30 minutes.

3. Media inoculation

This step is performed as described in example 1.

4. Media reading

This step is performed as described in example 1.

5. Results:

The medium A comprising ceftazidime and tobramycin allowed the inhibition of all wild strains and all Case Hn tested. Only BLSE positive strains were detected in this medium.

Medium B comprising ceftriaxone and cloxacillin allowed to inhibit all Case HN and all wild strains except P. aeruginosa Case Hn and wild, and only the majority of BLSE positive strains grew.

The medium C comprising cefpodoxime and di-cloxacillin allowed the inhibition of all wild strains and the

Most Case HN without affecting the growth of BLSE positive strains.

Claims (3)

5 10 fifteen twenty 25 30 35 40 Four. Five 1. Use of a culture medium to distinguish: • a first group of E. coli BLSE bacteria; • a second group of KESC BLSE bacteria; • a third group of bacteria not resistant to beta-lactamines; said culture medium comprising: • a first substrate that allows the detection of an enzymatic activity beta-glucuronidase or beta-galactosidase; • a second substrate that allows the detection of a beta-glucosidase or tryptophanase or deaminase activity; • cefpodoxime; • cloxacillin. 2. Use according to claim 1 of a culture medium to distinguish: • a first group of E. coli BLSE bacteria; • a second group of KESC BLSE bacteria; • a third group of bacteria, not resistant to beta-lactamines and / or cephalosporinases; • a fourth group of Proteeae BLSE bacteria; said culture medium comprising: • a first substrate that allows the detection of an enzymatic activity beta-glucuronidase or beta-galactosidase; • a second substrate that allows the detection of a beta-glucosidase activity; • cefpodoxime, • cloxacillin, • vancomycin, • amphotericin B, • a third substrate that allows the detection of a deaminase activity. 3. Use of a culture medium according to claim 2, said culture medium further comprising cefsulodine.